Dorothy Hodgkin: From Nobel Laureate to Google Doodle

http://www.google.com/doodles/dorothy-hodgkins-104th-birthday

The first time I heard of Dorothy Hodgkin was last May when Google featured her model of Penicillin as its doodle. Side note: I actually didn't see it myself, but was lucky enough to have a mother-in-law who was interested in my science and pointed it out to me. Having completed my dissertation in X-ray crystallography, I was surprised to not have heard of her before: the third woman to win the Nobel Prize in Chemistry for “her determinations by X-ray techniques of the structures of important biochemical substances (1).” The two other women were of course Marie Curie, and unbeknownst to me, her daughter, Irène Joliot-Curie. Dorothy Hodgkin determined the X-ray structures for important biomolecules, such as penicillin and Vitamin B12, as well as making her mark on protein crystallography. Her structure of Vitamin B12 allowed for the synthesis of the vitamin to treat megaloblastic anemia. I’ve written a separate post describing the field of X-ray crystallography in more detail here.

 Structure of Vitamin B12, one of the most complex structures that  had been determined by 

X-ray Crystallography at the time.  Image courtesy of chemicalheritage.org

It was apparent from my readings into her life that Dorothy Hodgkin is an example of the type of scientist I always hoped to be: committed, passionate, and steadfast about her work and its implications, well rounded and diverse in her interests, humble, yet confident in what she wanted in her life from an early age. Of course, sexism is not hard to find in her story. The headlines when she won the Nobel Prize in 1964 was "British woman wins Nobel Prize – £18,750 prize to mother of three." in the Telegraph and "Oxford housewife wins Nobel" in The Daily Mail. But she seemed to rise above it all.

Her father taught and worked in archaeology in Egypt, the Sudan, and Jerusalem and Dorothy would visit at different times in her life (2). She carried a love of Africa throughout her life, later making many trips to Ghana while her husband worked at the University of Ghana, and even for a time as advisor to the Ghanaian president (2,4). Eventually, her daughter would teach in Zambia (2).

Dorothy’s life is rife with the stuff of good dramas. According to a 1998 biography, her very influentional advisor, J.D. Bernal and she were lovers on and off for years, even after she married (3). The politics of this advisor, as well as her husband’s were of a communist persuasion and she was banned from travelling to the U.S. without a CIA waiver (4). It is unclear if she held the same political views but after being refused entry to the U.S. to attend a conference organized by Linus Pauling, she travelled to the Soviet Union, attempting to set up scientific collaborations. She also visited China many times throughout her career and had many collaborations with Chinese and Indian scientists. She campaigned for inclusion of countries into the International Union of Crystallography such as China and the Soviet Union, who were banned for political reasons (4).

Dorothy believed it was the responsibility of scientists to help make the world a better place. She was president of the Pugwash council from the mid 1970s to late 1980s (4). Never heard of the Pugwash conferences? Neither had I. Pugwash is an international organization established in the years following WWII, bringing together scholars with the goal towards international conflict resolution and nuclear and chemical weapon disarmament.

Dorothy Hodgkin, image courtesy of chemicalheritage.org

What drew me to her, more than her politics though was her dedication to solving a particular problem. She studied one protein, insulin for more than 35 years, eventually solving the structure. In her Nobel Lecture she recounts a sentence from the W.H. Braggs book “Concerning the Nature of Things” which peaked her interest in the field as a teenager, “”Broadly speaking, the discovery of X-rays has increased the keenness of our vision over ten thousand times and we can now ‘see’ the individual atoms and molecules.”” A few sentences later she writes, “The process of 'seeing' with X-rays was clearly more difficult to apply to such systems than my early reading of Bragg had suggested; it was with some hesitation that I began my first piece of research work with H. M. Powell…(5).” Four years after giving this lecture she finally determined the structure. When she originally set out in the 1930s, the technology and methodology was not advanced enough to study proteins. She continued to.. Her structure contributed to the knowledge of receptor/hormone binding and allowed for reliable synthesis of the hormone as a treatment for diabetes. 

This part of her story resonated with me as I remember seeing the beautiful pictures of protein structures in late bachelor studies and trying to wrap my mind around how one came to these pictures. Indeed, I would discover it was more complex than even I imagined. And I was lucky to benefit from all the amazing advances that semi-automate the steps along the way from setting up hundreds of crystal conditions (or in my case, thousands) to shooting the crystals with X-rays in California while sitting in my house in Baltimore to solving the structure using information gained by other crystallographers on similar proteins. Even with these advances, it took three and a half years to determine the structure of my one and only protein, Plasmodium falciparum Atg8.

It was with some sadness that I left the crystallography world behind, with its helpful and always willing to commiserate, yet highly critical, compatriots. My experiences make me appreciate all the more the contributions that Dorothy made in the then male-dominated and inchoate field of biomolecular and protein X-ray crystallography.

References:

1. (2014). Dorothy Crowfoot Hodgkin - Facts. Nobelprize.org. Nobel Media.

2. (2014). Dorothy Crowfoot Hodgkin – Biography. Nobelprize.org. Nobel Media AB 2014. Retrieved August 24, 2014. Retrieved from http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1964/hodgkin-bio.html.

3. Ferry, Georgina (1998). Dorothy Hodgkin: A Life. London: Granta Books.

4. Howard, J.A.K. (2003). Dorothy Hodgkin and her contributions to biochemistry. Nat Rev MCB. 4, 891-896.

5. Hodgkin D.C. (1964). The X-ray Analysis of complicated molecules. Nobel Lectures. Chemistry 1963-1970, Elsevier Publishing Company, Amsterdam, 1972.

A quick introduction into protein X-ray crystallography:

What information do we get from X-ray crystallography?

Proteins, which are the machines inside our cells that use energy to do countless tasks, such as copy our DNA for the next cell, cause our muscles to contract, bind hormones, allow our neurons to release hormones and neurotransmitters. It is vastly helpful to understand what proteins look like, their 3-dimensional structure, to understand how they work. For example, take the protein, DNA helicase. In order for our genome to be passed on to newly-divided cells or our progeny (in the form of sperm and egg cells), it must be copied. In order to be copied, it must be temporarily pulled apart from its more stable double-stranded form, to a single stranded-form where the information is accessible. DNA helicase is a motor that uses energy from ATP to unwind the DNA. In the image below, you can see that the structure of one molecule of the protein, which alone doesn’t really explain its function. But the protein binds five other identical helicase molecules to form a donut-shape, which encircles one strand of the DNA. The motor then drives the helicase forward, pulling the two strands apart by shear force.

 

 (PDB code: 2R5U) (http://www.pdb.org/pdb/explore/explore.do?structureId=2R5U)

This crystal structure is specifically of the helicase protein from the tuberculosis bacteria. Knowing the structure allows scientists to design drugs that bind the protein in the bacteria, but not in humans, and to prevent the bacteria from replicating.

How do we get this information?

Proteins are extremely small, too small to see with the human eye and the structure is too small to see with a microscope. But if we can get the protein to crystallize, forming a regularly repeating structure that is much bigger than the single protein alone, we amplify the signal-to-noise ratio. X-rays are then shot at the crystal. When the X-rays hit the atoms that make up the protein, they are diffracted, that is, the direction and intensity change. The pattern of diffraction provides information about the position of the atoms in the protein and the diffraction pattern is captured by a special camera device behind the protein. Of course, back when Dorothy Hodgkin was solving the structures of penicillin and Vitamin B12, there was no camera, the diffraction pattern was recorded on X-ray film. Using mathematical formulas, we can then backtrack to the structure, but there is a big caveat; some information, the phases, are missing from the diffraction pattern. There are several ways, which I won’t go into here, for crystallographers to get the missing information and solve the structure.

Dorothy Hodgkin contributed to the methods available to get this missing information and solve the phase problem. This included using selenium derivatives and using microorganisms to incorporate the selenium into the protein.

The what and why of the Ebola virus disease

 Electron micrograph of Ebola virus (Cynthia Goldsmith, CDC (wikimedia commons)

With the death toll surpassing one thousand, this is the largest Ebola outbreak since its emergence in 1976. The outbreak is unusual in its occurrence on the west coast of Africa rather than central and also in its spread to urban areas. The outbreak has received much news coverage in recent weeks as the death toll rises and two Americans infected were flown home to receive “experimental therapy.” Why does the outbreak capture the international attention so much more than diseases such as malaria, which kills three times that number…in one day? Perhaps one reason is that malaria is perceived as a chronic condition in developing countries, with eradication a far-fetched goal. The horrific effects of Ebola, hemorrhagic fever, internal bleeding and death resulting from multiple organ failure are probably even more of a contributor. This, combined with a mortality rate of up to 90% (though the numbers are closer to 60% in the current outbreak), makes for a terrifying disease. With the announcement by the WHO this week that experimental drugs can be used in Africa, it seemed like a good time to write a post about the biology behind the Ebola disease and how these much-discussed experimental drugs work. I apologize in advance if this information seems basic to readers in the scientific field, especially virologists, but this post is aimed at those less familiar with science.

The pathogen that causes Ebola is a virus. Viruses are different from bonafide living organisms in that they lack cellular structure and cannot make their own proteins. Proteins are the workhorses of the cell, molecular machines that replicate the genome, allow cells to divide, secrete, move, etc. But viruses are just genetic material, either RNA or DNA, enclosed in a protein coat and possibly a lipid envelope (depending on the type of virus). In order to make more of themselves, the virus must invade cells, be it cells of bacteria, plants, or mammals, and hijack the host machinery. The Ebola virus is a single-stranded negative sense RNA virus. This refers to what has to happen inside the host cell in order to replicate. Its genome is encoded in RNA, which, although very similar to DNA (used by most organisms to encode their genome), is chemically less stable than DNA. The virus has a helical protein coat that protects its genome, giving it a striking long filamentous appearance.

Journey of the Ebola virus:
The virus can spread to a new person when blood or body secretions containing the virus come in contact with mucus membranes or broken skin. A glycoprotein (made of a chain of amino acids and sugar) forms spikes on the virus surface and attach to the host cells. The viral proteins bind receptors on the host cell and trick the host cell into engulfing the virus, bringing it inside the cell (Saeed et al., 2010). The virus then needs to make a version of its genome that the cell can read like a blueprint to make more RNA genome, as well as the structural elements of the virus. Luckily for the virus, it has brought a protein, made during its previous infection to convert the genome into a proper blueprint. The newly copied and packaged viruses then must get out of the cell and invade new ones. The Ebola virus “buds” from the host cell, enclosing itself in lipids from the plasma membrane of the host cell, which is the barrier that separates cells from their environment. One protein in particular on the virus envelope is central to this step and is therefore being investigated as a potential drug target (Jasenosky et al., 2001, Timmons et al., 2001). However this is not what is in the ZMapp drug given to three Ebola patients.

Why is Ebola so deadly?
While some viruses infect only certain types of cells, the Ebola virus spreads through the blood system to infect cells in many organs. Patients often suffer liver, kidney, and other organ failure. A major contributor of the mortality of the disease is “immune paralysis” where patients are unable to mount a defense. This is because many of the proteins the virus makes are specialized to interfere with our defense systems allowing the pathogen to wreak havoc unchecked (Lubaki et al., 2013). For example, while our immune system is designed to sense viral RNA (double stranded RNA), a protein made by the Ebola virus covers and hides the RNA from the host. And any amount of interferon signaling activation that is achieved is inhibited later on by the same and other viral proteins (Leung et al., 2010). Additionally one of the first cells infected by the virus are macrophages, immune cells required early on to recruit other immune cells.  This both prevents them from doing their job and causes them to secrete substances that leads to the cell death of other immune cells and makes capillaries leaky, causing internal bleeding (Geisbert et al, 2003).

What’s in this experimental drug?
When infected by bacteria or viruses, our immune systems produce antibodies, “Y”-shaped proteins that bind a protein on the foreign invader similarly to a lock and key mechanism, using the two tips of the “Y”. This neutralizes the pathogen and recruits other parts of the immune system. Therefore antibodies against Ebola virus injected into a patient could help fight the infection.

But where can we get these antibodies?
Mice similarly produce antibodies so scientists at Mapp Biopharmaceutical injected mice with the virus and harvested the antibodies the mice produced. After identifying the antibody reactive against Ebola, they modified the genetic blueprint encoding the antibody so it looked more human-like. This is to prevent an immune reaction in humans, in which the immune system would see the injected antibody as a foreign invader rather than a helping hand.

How to make large amounts of this antibody? 
Since all organisms use the same genetic code, scientists can make all kinds of cells produce the protein of their choice. In graduate school, I made E. coli bacteria produce proteins from the malaria parasite so that I could study the protein more easily (hence my comment about the deadliness of malaria in the introduction). However since bacteria aren’t very good at making complex antibodies, mammalian cell lines are often used instead. Mapp Biopharmaceutical uses tobacco plants because production is faster and cheaper. The DNA is inserted into the plant cells and the plant’s machinery makes the recombinant protein, which is then harvested. ZMapp, the serum that was used on the two Americans, is a combination of antibodies made in this way. It is important to keep in mind that although the two American patients appear to be responding positively to ZMapp, it is impossible to determine from this that the drug works. The only way to determine the effectiveness of a drug is through clinical trials in which a control group receives a placebo. However with both the ethical questions of testing drugs in developing countries and the extremely short supply of serum, clinical trials are unlikely to occur in the near future.

References:
Geisbert, T., et al. (2003). Pathogenesis of Ebola Hemorrhagic Fever in Cynomolgus Macaques. Am J Pathol. 163, 2347-2370.

Feldmann H., et al. (2001). Biosynthesis and role of filoviral glycoproteins. J Gen Virol. 82, 2839-48.

Jasenosky, L. D., Neumann, G., Lukashevich, I., and Kawaoka, Y. (2001). Ebola virus VP40-induced particle formation and association with the lipid bilayer. J Virol. 75, 5205–5214.

Lubaki, N.M., et al. (2013). The Lack of Maturation of Ebola Virus-Infected Dendritic Cells Results from the Cooperative Effect of at Least Two Viral Domains. J Virol. 87, 12506.

Leung, D.W., et al. (2010). Structural basis for dsRNA recognition and interferon antagonism by Ebola VP35. Nat Struct and Mol Biol. 17, 165–172.

Timmins, J., Scianimanico, S., Schoehn, G., and Weissenhorn, W. (2001). Vesicular release of ebola virus matrix protein VP40. Virol. 283, 1–6.

Saeed, M. F., et al. (2010). Cellular Entry of Ebola Virus Involves Uptake by a Macropinocytosis-Like Mechanism and Subsequent Trafficking through Early and Late Endosomes. Plos Path. 6, 1-15.

Wahl-Jensen, V., et al. (2005). Effects of Ebola Virus Glycoproteins on Endothelial Cell Activation and Barrier Function. J Virol. 79, 10442-10450.